Promoters for regulation of gene expression in plant roots

ABSTRACT

The present invention is directed to promoters isolated from maize and functional equivalents thereto. The promoters of the present invention have particular utility in driving root-specific expression of heterologous genes that impart increased agronomic, horticultural and/or pesticidal characteristics to a given promoters of the invention and transformed plant tissues containing DNA molecules comprising a promoter of the invention operably linked to a heterologous gene or genes, and seeds thereof.

This is a national stage application under 35 U.S.C. §371 ofInternational Application No. PCT/US02/35374, filed on Nov. 4, 2002,which is entitled to the benefit of U.S. Provisional Application No.60/337,026, filed on Nov. 7, 2001, both of which are incorporated hereinby reference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to the field of plant molecularbiology and the regulation of gene expression in plants. Morespecifically, the present invention relates to the regulation of geneexpression in plant roots.

BACKGROUND OF THE INVENTION

Manipulation of crop plants to alter and/or improve phenotypiccharacteristics (such as productivity or quality) requires theexpression of heterologous genes in plant tissues. Such geneticmanipulation has become possible by virtue of two discoveries: theability to transform heterologous genetic material into a plant cell andby the existence of promoters that are able to drive the expression ofthe heterologous genetic material.

It is advantageous to have the choice of a variety of differentpromoters so as to give the desired effect(s) in the transgenic plant.Suitable promoters may be selected for a particular gene, construct,cell, tissue, plant or environment. Promoters that are useful for planttransgene expression include those that are inducible, viral, synthetic,constitutive (Odell et al., 1985, Nature 313: 810-812; Granger & Cyr,2001, Plant Cell Repo. 20: 227-234), temporally regulated, spatiallyregulated, tissue-specific, and spatio-temporally regulated (Kuhlemeieret al. 1987, Ann. Rev. Plant Physiol. Plant Mol. Biol. 38: 221-257).Promoters from bacteria, fungi, viruses and plants have been used tocontrol gene expression in plant cells.

Promoters consist of several regions that are necessary for fullfunction or the promoter. Some of these regions are modular, in otherwords they can be used in isolation to confer promoter activity or theymay be assembled with other elements to construct new promoters. Thefirst of these promoter regions lies immediately upstream of the codingsequence and forms the “core promoter region” containing consensussequences, normally 20-70 base pairs immediately upstream of the codingsequence. The core promoter region contains a TATA box and often aninitiator element as well as the initiation site. The precise length ofthe core promoter region is not fixed but is usually well recognizable.Such a region is normally present, with some variation, in mostpromoters. The base sequences lying between the variouswell-characterized elements appear to be of lesser importance. The corepromoter region is often referred to as a minimal promoter regionbecause it is functional on its own to promote a basal level oftranscription.

The presence of the core promoter region defines a sequence as being apromoter: if the region is absent, the promoter is non-functional. Thecore region acts to attract the general transcription machinery to thepromoter for transcription initiation. However, the core promoter regionis insufficient to provide fill promoter activity. A series ofregulatory sequences, often upstream of the core, constitute theremainder of the promoter. The regulatory sequences determine expressionlevel, the spatial and temporal pattern of expression and, for a subsetof promoters, expression under inductive conditions (regulation byexternal factors such as light, temperature, chemicals and hormones).Regulatory sequences may be short regions of DNA sequence 6-100 basepairs that define the binding sites for trans-acting factors, such astranscription factors. Regulatory sequences may also be enhancers,longer regions of DNA sequence that can act from a distance from thecore promoter region, sometimes over several kilobases from the coreregion. Regulatory sequence activity may be influenced by trans-actingfactors including general transcription machinery, transcription factorsand chromatin assembly factors.

Frequently, it is desirable to have tissue-specific expression of a geneof interest in a plant. Tissue-specific promoters promote expressionexclusively in one set of tissues without expression throughout theplant; tissue-preferred promoters promote expression at a higher levelin a subset of tissues with significantly less expression in the othertissues of the plant. For example, one may desire to express avalue-added product only in corn seed but not in the remainder of theplant. Another example is the production of male sterility bytissue-specific ablation. In this case, a phytotoxic product isexpressed only in the male tissue of the plant to ablate that specifictissue while other tissues of the flower as well as the rest of theplant remain intact. Many aspects of agricultural biotechnology use andrequire tissue-specific expression.

One important example of a need for promoters is for the expression ofselected genes in plant roots. The plant root consists of many celltypes such as epidermal, root cap, columella, cortex, pericycle,vascular and root hair forming trichoblasts, organized into tissues orregions of the root, for example, the root tip, root epidermis,meristematic zone, primary root, lateral root, root hair, and vasculartissue. Promoters isolated as root-specific or root-preferred can bebiased towards promotion of expression in one or a few of these celltypes. This cell-specific activity can be useful for specificapplications such as regulating meristematic activity in only themeristematic cell zone or expression of A nematicidal gene in only thecell types that are contacted by the nematode pest. In other cases,broader cell-type specificity may be desired to express genes ofinterest throughout the root tissue. This may be useful in expressing aninsecticidal gene to control an insect pest that feeds on plant roots,for instance corn rootworm (Diabrotica spp.). Broader cell-type rootspecificity may be accomplished with a single root-specific promoterwith broad cell-type specificity or by using two or more root-specificor root-preferred promoters of different cell-type specificities forexpression. A limited number of examples of root-preferred androot-specific promoters have been described. These include the RB7promoter from Nicotiana tabacum (U.S. Pat. Nos. 5,459,252 and5,750,386); the ARSK1 promoter from Arabidopsis thaliana (Hwang andGoodman (1995) Plant J 8:37-43), the MR7 promoter from Zea mays (U.S.Pat. No. 5,837,848), the ZRP2 promoter of Zea mays (U.S. Pat. No.5,633,363), and the MTL promoter from Zea mays (U.S. Pat. Nos. 5,466,785and 6,018,099). Many of these examples disclose promoters withexpression patterns confined to a limited number of root tissues. Othersfail to provide the root-specificity needed for expression of selectedgenes. Thus, there is a need in the art for isolation andcharacterization of new root promoters to obtain those of differentbreadth, expression level and specificity of cell-type expression forroot-specific and root-preferred expression, particularly forroot-specific expression.

SUMMARY OF THE INVENTION

Within the present invention, compositions and methods for directingroot-specific expression in transgenic plants are provided. Inparticular, novel nucleic acid molecules isolated from Zea mays, thatdrive expression of heterologous genes in a root-specific manner inplants, are provided. The invention is further drawn to expressioncassettes and vectors comprising the novel nucleic acid molecules of theinvention operably linked to heterologous coding sequences. Theinvention is still further drawn to transgenic plants comprising theexpression cassettes of the invention. The present invention alsoprovides methods for specifically expressing a heterologous codingsequence in transgenic plant roots, for isolating a root-specific cDNA,for isolating a nucleic acid molecule useful for directing root-specificexpression and for isolating a root-specific promoter. The inventionfurther provides primers and nucleic acid probes to identify relatednucleotide sequences from other plant genomes that direct root-specificor root-preferred transcription.

According to one aspect, the present invention provides an isolatednucleic acid molecule which codes for a promoter capable of directingroot-specific transcription in a plant, wherein the nucleotide sequenceof the promoter comprises a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3, or SEQ ID NO: 4; (b) a nucleotide sequence that hybridizesunder high stringency conditions to a nucleotide sequence of a); and (c)a nucleotide sequence comprising a fragment of a sequence of (a),wherein the fragment maintains function of the nucleotide sequence of(a).

In another aspect, the present invention provides fragments of thenucleotide sequences set forth in SEQ ID NOS: 1-4 wherein the fragmentscode for promoters capable of directing root-specific transcription in aplant. In a preferred embodiment of this aspect, the fragments are madeby making 5′-deletions in the nucleotide sequences set forth in SEQ IDNOS: 1-4. More preferably the fragment is a 5′-deletion of thenucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

The present invention also provides an expression cassette comprisingthe nucleic acid molecule of the invention operably linked to aheterologous coding sequence. In one embodiment, the expression cassettecomprises a heterologous coding sequence selected from the groupconsisting of insecticidal coding sequences, nematicidal codingsequences, herbicide-tolerance coding sequences, anti-microbial codingsequences, anti-fungal coding sequences, anti-viral coding sequences,abiotic stress tolerance coding sequences, nutritional quality codingsequences, visible marker coding sequences and selectable marker codingsequences. In a preferred embodiment, the expression cassette comprisesan insecticidal coding sequence that encodes a toxin active against acoleopteran pest. In a preferred aspect of this embodiment, thecoleopteran pest is a species in the genus Diabrotica. In yet anotherembodiment, the expression cassette comprises an abiotic stresstolerance coding sequence including but not limited to drought stress,nutrient stress, salt stress, water stress and heavy metal stress. Instill another embodiment, the expression cassette comprises a visiblemarker coding sequence including but not limited to green fluorescentprotein (GFP), β-glucuronidase (GUS), and luciferase (LUC). In yetanother embodiment, the expression cassette comprises a selectablemarker coding sequence including but not limited to phosphomannoseisomerase (PMI), an antibiotic resistance gene such as hygromycin,kanamycin and the like, a herbicide tolerance gene such asphosphinothricin and the like and bamase (bar).

The present invention also provides a recombinant vector comprising theexpression cassette of the invention. In a preferred embodiment, therecombinant vector is a plasmid.

Further, the present invention provides a transgenic non-human host cellcomprising the expression cassette of the invention. A transgenic hostcell according to this aspect of the invention is preferably a plantcell. Even further, the present invention provides a transgenic plantcomprising such a transgenic plant cell. A transgenic plant according tothis aspect of the invention may be sorghum, wheat, sunflower, tomato,cole crops, cotton, rice, soybean, sugar beet, sugarcane, tobacco,barley, oilseed rape and maize, preferably maize or rice. Still further,the present invention provides transgenic seed from the group oftransgenic plants consisting of sorghum, wheat, sunflower, tomato, colecrops, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley,oilseed rape and maize. In a particularly preferred embodiment, thetransgenic seed is from a transgenic maize plant or rice plant.

In another aspect, the present invention provides a method ofspecifically expressing a heterologous coding sequence in transgenicplant roots under transcriptional control of a nucleic acid molecule ofthe invention, comprising: (a) transforming plant cells with a vectorwherein the vector comprises the nucleic acid molecule of the inventionoperably linked to a heterologous coding sequence, (b) growing thetransgenic plant cells comprising the vector, and (c) producingtransgenic plants from the transformed plant cells wherein theheterologous coding sequence is specifically expressed in plant rootsunder control of a nucleic acid molecule of the invention. In oneembodiment of this aspect, the transgenic plant is a maize plant or arice plant. In another embodiment of this aspect, the heterologouscoding sequence is selected from the group consisting of insecticidalcoding sequences, nematicidal coding sequences, herbicide tolerancecoding sequences, anti-microbial coding sequences, anti-fungal codingsequences, anti-viral coding sequences, abiotic stress tolerance codingsequences, nutritional quality coding sequences, visible marker codingsequences and selectable marker coding sequences. In yet anotherembodiment, the invention provides transgenic plants produced accordingto this aspect. In a preferred embodiment the transgenic plants aremaize plants or rice plants.

In a further aspect, the present invention provides a method ofisolating a promoter capable of directing root-specific expression inplants comprising: (a) preparing a nucleic acid probe from any one ofSEQ ID NOS: 1-4; (b) hybridizing the nucleic acid probe to either cDNAor genomic DNA prepared from a plant; and (c) isolating a hybridizingsequence from the cDNA or the genomic DNA with at least 70% identity tothe nucleic acid probe.

In another aspect, the present invention provides a method ofidentifying fragments of a promoter capable of directing root-specificexpression in plants comprising: (a) providing the isolated promotersequence according to the invention; (b) generating fragments of thepromoter sequence of step (a); (c) transforming plants with thefragments of step (b) operably linked to a heterologous coding sequence;and (d) identifying the fragments of step (b) having promoter activityin a transgenic plant by expression of the heterologous coding sequence.

Also provided by the present invention is a primer comprising at least16 contiguous nucleotides of any one of SEQ ID NOS: 1-4. Further, thepresent invention provides a hybridization probe comprising at least 50contiguous nucleotides of any one of SEQ ID NOS: 1-4.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NO: 1 is the nucleotide sequence of the MRS1 promoter.

SEQ ID NO: 2 is the nucleotide sequence of the MRS2 promoter.

SEQ ID NO: 3 is the nucleotide sequence of the MRS3 promoter.

SEQ ID NO: 4 is the nucleotide sequence of the MRS4 promoter.

SEQ ID NO: 5 is the sequence of the root-specific cDNA designated 22P8.

SEQ ID NO: 6 is the predicted amino acid sequence encoded by SEQ ID NO:5.

SEQ ID NO: 7 is the sequence of the root-specific cDNA designated 10B21.

SEQ ID NO: 8 is the predicted amino acid sequence encoded by SEQ ID NO:7.

SEQ ID NO: 9 is the sequence of the root-specific cDNA designated 2D14.

SEQ ID NO: 10 is the predicted amino acid sequence encoded by SEQ ID NO:9.

SEQ ID NO: 11 is the sequence of the root-specific cDNA designated 4H19.

SEQ ID NO: 12 is the predicted amino acid sequence encoded by SEQ ID NO:11.

SEQ ID NOS: 13-16 are primers useful in isolating 22P8 cDNA and relatedsequences.

SEQ ID NOS: 17-20 are primers useful in isolating 10B21 cDNA and relatedsequences.

SEQ ID NOS: 21-22 are primers useful in isolating 2D14 cDNA and relatedsequences.

SEQ ID NOS: 23-26 are primers useful in isolating 4H19 cDNA and relatedsequences.

SEQ ID NO: 27 is an adapter primer useful according to the presentinvention.

SEQ ID NO: 28 is a primer useful in isolating the 22P8 promoter andrelated sequences.

SEQ ID NOS: 29-30 are primers useful in isolating the 10B21 promoter andrelated sequences.

SEQ ID NOS: 31-32 are primers useful in isolating the 2D14 promoter andrelated sequences.

SEQ ID NOS: 33-34 are primers useful in isolating the 4H19 promoter andrelated sequences.

SEQ ID NO: 35 is an adapter primer useful according to the presentinvention.

SEQ ID NO: 36 is the 22P8GSP7 forward primer.

SEQ ID NO: 37 is a 5′λ arm primer.

SEQ ID NO: 38 is a 3′λ arm primer.

SEQ ID NOS: 39-40 are primers useful in amplifying the MRS1L promoter.

SEQ ID NOS: 41-42 are primers useful in adding att sites to the MRS1Lpromoter.

SEQ ID NOS: 43-44 are primers useful in adding attB1 and attB2 sites tothe 5′ and 3′ ends of the promoters of the present invention.

SEQ ID NO: 45 is a primer useful in amplifying the MRS1S promoter.

SEQ ID NO: 46 is a primer useful adding att sites to the MRS1S promoter.

SEQ ID NOS: 47-50 are primers useful in amplifying the MRS2 promoter.

SEQ ID NOS: 51-52 are primers useful in adding att sites to the MRS2promoter.

SEQ ID NOS: 53-54 are primers useful in amplifying the MRS3 promoter.

SEQ ID NOS: 55-56 are primers useful in adding att sites to the MRS3promoter.

SEQ ID NOS: 57-59 are primers useful in amplifying the MRS4 promoter.

SEQ ID NOS: 60-61 are primers useful in adding att sites to the MRS4promoter.

SEQ ID NOS: 62-63 are primers useful in constructing a binarydestination vector according to the present invention.

SEQ ID NOS: 64-67 are primers useful in isolating the MRS2M and MRS2Spromoters.

DEFINITIONS

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of protein from anendogenous gene or a transgene.

“Chimeric” is used to indicate that a DNA sequence, such as a vector ora gene, is comprised of two or more DNA sequences of distinct originthat are fused together by recombinant DNA techniques resulting in a DNAsequence, which does not occur naturally.

“Chromosomally-integrated” refers to the integration of a foreign geneor DNA construct into the host DNA by covalent bonds. Where genes arenot “chromosomally integrated” they may be “transiently expressed.”Transient expression of a gene refers to the expression of a gene thatis not integrated into the host chromosome but functions independently,either as part of an autonomously replicating plasmid or expressioncassette, for example, or as part of another biological system such as avirus.

“Coding sequence” refers to a DNA or RNA sequence that codes for aspecific amino acid sequence and excludes the non-coding sequences. Itmay constitute an “uninterrupted coding sequence”, i.e., lacking anintron, such as in a cDNA or it may include one or more introns boundedby appropriate splice junctions. An “intron” is a sequence of RNA whichis contained in the primary transcript but which is removed throughcleavage and re-ligation of the RNA within the cell to create the matureMRNA that can be translated into a protein.

“Constitutive promoter” refers to a promoter that is able to express thegene that it controls in all or nearly all of the plant tissues duringall or nearly all developmental-stages of the plant, thereby generating“constitutive expression” of the gene.

“Co-suppression” and “sense suppression” refer to the production ofsense RNA transcripts capable of suppressing the expression of identicalor substantially identical transgene or endogenous genes (U.S. Pat. No.5,231,020).

“Contiguous” is used herein to mean nucleic acid sequences that areimmediately preceding or following one another.

“Corn rootworm” or “corn rootworms”, as used herein, refer to insects ofthe genus Diabrotica, including the southern corn rootworm, the northerncorn rootworm, the western corn rootworm, and the Mexican corn rootwormeither in the larval or adult stage, preferably in the larval stage. Theroot-specific promoters of the invention are used to express cornrootworm toxins in the roots of transgenic plants thus protecting fieldsof transgenic plants from corn rootworm damage. The term “corn rootworm”and Diabrotica are herein used interchangeably.

“Expression” refers to the transcription and stable accumulation ofmRNA. Expression may also refer to the production of protein.

“Expression cassette” as used herein means a DNA sequence capable ofdirecting expression of a particular nucleotide sequence in anappropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The coding region usually codesfor a protein of interest but may also code for a functional RNA ofinterest, for example antisense RNA or a nontranslated RNA, in the senseor antisense direction. The expression cassette comprising thenucleotide sequence of interest may be chimeric, meaning that at leastone of its components is heterologous with respect to at least one ofits other components.

The “expression pattern” of a promoter (with or without an enhancer) isthe pattern of expression level that shows where in the plant and inwhat developmental stage the promoter initiates transcription.Expression patterns of a set of promoters are said to be complementarywhen the expression pattern of one promoter shows little overlap withthe expression pattern of the other promoter.

“Gene” refers to a nucleic acid fragment that expresses mRNA, functionalRNA, or specific protein, including regulatory sequences. The term“Native gene” refers to a gene as found in nature. The term “chimericgene” refers to any gene that contains 1) DNA sequences, includingregulatory and coding sequences, that are not found together in nature,or 2) sequences encoding parts of proteins not naturally adjoined, or 3)parts of promoters that are not naturally adjoined. Accordingly, achimeric gene may comprise regulatory sequences and coding sequencesthat are derived from different sources, or comprise regulatorysequences and coding sequences derived from the same source, butarranged in a manner different from that found in nature. A “transgene”refers to a gene that has been introduced into the genome bytransformation and is stably maintained. Transgenes may include, forexample, genes that are either heterologous or homologous to the genesof a particular plant to be transformed. Additionally, transgenes maycomprise native genes inserted into a non-native organism, or chimericgenes. The tern “endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism but one that is introduced intothe organism by gene transfer.

“Gene silencing” refers to homology-dependent suppression of viralgenes, transgenes, or endogenous nuclear genes. Gene silencing may betranscriptional, when the suppression is due to decreased transcriptionof the affected genes, or post-transcriptional, when the suppression isdue to increased turnover (degradation) of RNA species homologous to theaffected genes. (English, et al., 1996, Plant Cell 8:179-1881). Genesilencing includes virus-induced gene silencing (Ruiz et al., 1998,Plant Cell 10:937-946).

“Genetically stable” and “heritable” refer to chromosomally-integratedgenetic elements that are stably maintained in the plant and stablyinherited by progeny through successive generations.

“Heterologous DNA Sequence” is a DNA sequence not naturally associatedwith a host cell into which it is introduced, including non-naturallyoccurring multiple copies of a naturally occurring DNA sequence.

“Inducible promoter” refers to those regulated promoters that can beturned on in one or more cell types by an external stimulus, such as achemical, light, hormone, stress, or a pathogen.

“Insecticidal” is defined as a toxic biological activity capable ofcontrolling insects, preferably by killing them.

“5′ non-coding sequence” refers to a nucleotide sequence located 5′(upstream) to the coding sequence. It is present in the fully processedmRNA upstream of the initiation codon and may affect processing of theprimary transcript to mRNA, mRNA stability or translation efficiency.(Turner et al., 1995, Molecular Biotechnology, 3:225).

“3′ non-coding sequence” refers to nucleotide sequences located 3′(downstream) to a coding sequence and include polyadenylation signalsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the MRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989, PlantCell, 1:671-680,).

The term “nucleic acid” refers to a polynucleotide of high molecularweight which can be single-stranded or double-stranded, composed ofmonomers (nucleotides) containing a sugar, phosphate and a base which iseither a purine or pyrimidine. A “nucleic acid fragment” is a fractionof a given nucleic acid molecule. In higher plants deoxyribonucleic acid(DNA) is the genetic material while ribonucleic acid (RNA) is involvedin the transfer of information contained within DNA into proteins. A“genome” is the entire body of genetic material contained in each cellof an organism. The term “nucleotide sequence” refers to a polymer ofDNA or RNA which can be single- or double-stranded, optionallycontaining synthetic, non-natural or altered nucleotide bases capable ofincorporation into DNA or RNA polymers.

The terms “open reading frame” and “ORF” refer to the amino acidsequence encoded between translation initiation and termination codonsof a coding sequence. The terms “initiation codon” and “terminationcodon” refer to a unit of three adjacent nucleotides (‘codon’) in acoding sequence that specifies initiation and chain termination,respectively, of protein synthesis (mRNA translation).

“Operably-linked” and “Operatively-linked” refer to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably-linked with a coding sequence or functional RNA when it iscapable of affecting the expression of that coding sequence orfunctional RNA (i.e., that the coding sequence or functional RNA isunder the transcriptional control of the promoter). Coding sequences insense or antisense orientation can be operably-linked to regulatorysequences.

“Overexpression” refers to the level of expression in transgenicorganisms that exceeds levels of expression in normal or untransformedorganisms.

“Plant tissue” includes differentiated and undifferentiated tissues orplants, including but not limited to roots, stems, shoots, leaves,pollen, seeds, tumor tissue and various forms of cells and culture suchas single cells, protoplast, embryos, and callus tissue. The planttissue may be in plants or in organ, tissue or cell culture.

“Preferred expression” is the expression of gene products that arepreferably expressed at a higher level in one or a few plant tissues(spatial limitation) and/or to one or a few plant developmental stages(temporal limitation) while in other tissues/developmental stages thereis a relatively low level of expression.

“Primary transformant” and “T0 generation” refer to transgenic plantsthat are of the same genetic generation as the tissue that was initiallytransformed (i.e., not having gone through meiosis and fertilizationsince transformation). “Secondary transformnants” and the “T1, T2, T3,etc. generations” refer to transgenic plants derived from primarytransformnants through one or more meiotic and fertilization cycles.They may be derived by self-fertilization of primary or secondarytransformants or crosses of primary or secondary transformants withother transformed or untransformed plants.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

“Promoter” refers to a nucleotide sequence, usually upstream (5′) to itscoding sequence, which controls the expression of the coding sequence byproviding the recognition for RNA polymerase and other factors requiredfor proper transcription. “Promoter regulatory sequences” consist ofproximal and more distal upstream elements, the latter elements oftenreferred to as enhancers. Accordingly, an “enhancer” is a DNA sequencethat can stimulate promoter activity and may be an innate element of thepromoter or a heterologous element inserted to enhance the level ortissue specificity of a promoter. It is capable of operating in bothorientations (normal or flipped), and is capable of functioning evenwhen moved either upstream or downstream from the promoter. Promotersmay be derived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven be comprised of synthetic DNA segments. A “minimal or corepromoter” is a promoter consisting only of all basal elements needed fortranscription initiation, such as a TATA-box and/or initiator.

“Reference sequence” as used herein is defined as a sequence that isused as a basis for sequence comparison. A reference sequence may be asubset or the entirety of a specified sequence; for example, as afragment of a full-length cDNA or gene sequence, or the full-length cDNAor gene sequence.

“Regulated promoter” refers to promoters that direct gene expression notconstitutively, but in a temporally- and/or spatially-regulated manner,and include both tissue-specific and inducible promoters. It includesnatural and synthetic sequences as well as sequences which may be acombination of synthetic and natural sequences. Different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions.

“Regulatory sequences” and “suitable regulatory sequences” each refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences includeenhancers, promoters, translation leader sequences, introns, andpolyadenylation signal sequences. They include natural and syntheticsequences as well as sequences which may be a combination of syntheticand natural sequences.

The term “RNA transcript” refers to the product resulting from RNApolymerase catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived by posttranscriptional processing of the primary transcript andis referred to as the mature RNA. “Messenger RNA” (MRNA) refers to theRNA that is without introns and that can be translated into protein bythe cell. “cDNA” refers to a single- or a double-stranded DNA that iscomplementary to and derived from MRNA. A “functional RNA” refers to anantisense RNA, ribozyme, or other RNA that is not translated. (butparticipates in a reaction or process as an RNA).

A “selectable marker gene” refers to a gene whose expression in a plantcell gives the cell a selective advantage. The selective advantagepossessed by the cells transformed with the selectable marker gene maybe due to their ability to grow in presence of a negative selectiveagent, such as an antibiotic or a herbicide, compared to the ability togrow of non-transformed cells. The selective advantage possessed by thetransformed cells may also be due to their enhanced capacity, relativeto non-transformed cells, to utilize an added compound as a nutrient,growth factor or energy source. A selective advantage possessed by atransformed cell may also be due to the loss of a previously possessedgene in what is called “negative selection”. In this, a compound isadded that is toxic only to cells that did not lose a specific gene (anegative selectable marker gene) present in the parent cell (typically atransgene).

“Specific expression” is the expression of gene products that is limitedto one or a few plant tissues (spatial limitation) and/or to one or afew plant developmental stages (temporal limitation).

Substantially identical: the phrase “substantially identical,” in thecontext of two nucleic acid or protein sequences, refers to two or moresequences or subsequences that have at least 60%, preferably 80%, morepreferably 90, even more preferably 95%, and most preferably at least99% nucleotide or amino acid residue identity, when compared and alignedfor maximum correspondence, as measured using one of the followingsequence comparison algorithms or by visual inspection. Preferably, thesubstantial identity exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably the sequences aresubstantially identical over at least about 150 residues. In anespecially preferred embodiment, the sequences are substantiallyidentical over the entire length of the coding regions. Furthermore,substantially identical nucleic acid or protein sequences performsubstantially the same function.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters. Those of skill in the art understand thatto avoid a high similarity to a reference sequence due to inclusion ofgaps in the polynucleotide sequence a gap penalty is typicallyintroduced and is subtracted from the number of matches.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, 1981, Adv. Appl. Math2: 482, by the homology alignment algorithm of Needleman & Wunsch, 1970,J. Mol. Biol. 48: 443, by the search for similarity method of Pearson &Lipman, 1988, Proc. Nat'l. Acad. Sci. 85: 2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Ausubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., 1990). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always>0) and N penalty score for mismatching residues;always<0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a word length (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989, Proc. Natl.Acad. Sci. 89: 10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90: 5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a test nucleicacid sequence is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test nucleic acidsequence to the reference nucleic acid sequence is less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001.

For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity to the promotersequences disclosed herein is preferably made using the BlastN program(version 1.4.7 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by the preferred program.

Another indication that two nucleic acid sequences are substantiallyidentical is that the two molecules hybridize to each other understringent hybridization conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent hybridizationconditions when that sequence is present in a complex mixture (e.g.,total cellular) of DNA or RNA. “Bind(s) substantially” refers tocomplementary hybridization between a probe nucleic acid and a targetnucleic acid and embraces minor mismatches that can be accommodated byreducing the stringency of the hybridization media to achieve thedesired detection of the target nucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology-Hybridization withNucleic Acid Probes part I chapter 2, “Overview of principles ofhybridization and the strategy of nucleic acid probe assays”, Elsevier,N.Y. Generally, high stringency hybridization and wash conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. Typically,under high stringency conditions a probe will hybridize to its targetsubsequence, but to no other sequences.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very high stringency conditions are selected to be equal to theT_(m) for a particular probe. An example of high stringencyhybridization conditions for hybridization of complementary nucleicacids which have more than 100 complementary residues on a filter in aSouthern or northern blot is 50% formamide with 1 mg of heparin at 42°C., with the hybridization being carried out overnight. An example ofvery high stringency wash conditions is 0.1 5M NaCl at 72° C. for about15 minutes. An example of high stringency wash conditions is a 0.2×SSCwash at 65° C. for 15 minutes (see, Sambrook, infra, for a descriptionof SSC buffer). Often, a high stringency wash is preceded by a lowstringency wash to remove background probe signal. An example mediumstringency wash for a duplex of, e.g., more than 100 nucleotides, is1×SSC at 45° C. for 15 minutes. An example low stringency wash for aduplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15minutes. For short probes (e.g., about 10 to 50 nucleotides), highstringency conditions typically involve salt concentrations or less thanabout 1.0 M Nn ion, typically about 0.01 to 1.0 M Na ion concentration(or other salts) at pH 7.0 to 8.3, and the temperature is typically atleast about 30° C. High stringency conditions can also be achieved withthe addition of destabilizing agents such as formamide. In general, asignal to noise ratio of 2× (or higher) than that observed for anunrelated probe in the particular hybridization assay indicatesdetection of a specific hybridization. Nucleic acids that do nothybridize to each other under high stringency conditions are stillsubstantially identical if the proteins that they encode aresubstantially identical. This occurs, for example, when a copy of anucleic acid is created using the maximum codon degeneracy permitted bythe genetic code.

Low stringency conditions include hybridization with a buffer solutionof 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium,citrate) at 50 to 55° C. Exemplary moderate stringency conditionsinclude hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary highstringency conditions include hybridization in 0% formamide, 1 M NaCl,1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.

The following are examples of sets of hybridization/wash conditions thatmay be used to clone homologous nucleotide sequences that aresubstantially identical to reference nucleotide sequences of the presentinvention: a reference nucleotide sequence preferably hybridizes to thereference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C.,more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirablystill in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C. with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,0.1% SDS at 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and, temperature of the finalwash solution. For DNA-DNA hybrds, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl Anal. Biochem. 138:267-284 (1984); TM81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)-500/L; where M is themolarity of monovalent cations, % GC is the percentage or guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The TM is the temperature (under defined ionic strength and pH)at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. T is reduced by about 1° C. for each 1% ofmismatching; thus, TM, hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with >90% identity are sought, the T_(m) can be decreased10° C. Generally, high stringency conditions are selected to be about19° C. lower than the thermal melting point (T_(m)) for the specificsequence and its complement at a defined ionic strength and pH. However,very high stringency conditions can utilize a hybridization and/or washat 1, 2, 3, or 4° C. lower than the thermal melting point (T_(m));moderately stringent conditions can utilize a hybridization and/or washat 6, 7, 8, 9, or 10° C. lower than the thermal melting point (T_(m));low stringency conditions can utilize a hybridization and/or wash at 11,12, 13, 14, 15, or 20° C. lower than the thermal melting point (T_(m)).Using the equation, hybridization and wash compositions, and desired T,those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T of lessthan 45° C. (aqueous solution) or 32° C. (formamide solution), it ispreferred to increase the SSC concentration so that a higher temperaturecan be used. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology-Hybridization with Nucleic Acid Probes, Part 1,Chapter 2 (Elsevier, N.Y.); and Ausubel et al., eds. (1995) CurrentProtocols in Molecular Biology, Chapter 2 (Greene Publishing andWiley—Interscience, N.Y.). Sec Sambrook et al. (1989) Molecular Cloning:A Laborator Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

A further indication that two nucleic acid sequences or proteins aresubstantially identical is that the protein encoded by the first nucleicacid is immunologically cross reactive with, or specifically binds to,the protein encoded by the second nucleic acid. Thus, a protein istypically substantially identical to a second protein, for example,where the two proteins differ only by conservative substitutions.

“Tissue-specific promoter” refers to regulated promoters that are notexpressed in all plant cells but only in one or more cell types inspecific organs (such as leaves, roots or seeds), specific tissues (suchas embryo or cotyledon), or specific cell types (such as leaf parenchymaor seed storage cells). These also include promoters that are temporallyregulated, such as in early or late embryogenesis, during fruit ripeningin developing seeds or fruit, in fully differentiated leaf, or at theonset of senescence.

“Transactivating gene” refers to a gene encoding a transactivatingprotein. It can encode a transcription factor. It can be a natural gene,for example, a plant transcriptional activator, or a chimeric gene, forexample, when plant regulatory sequences are operably-linked to the openreading frame of a transcription factor from another organism.“Transactivating genes” may be chromosomally integrated or transientlyexpressed. “Trans-activation” refers to switching on of gene by theexpression of another (regulatory) gene in trans.

A “transcriptional cassette” will comprise in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, aDNA sequence of interest, and a transcriptional and translationaltermination region functional in plants. The termination region may benative with the transcriptional initiation region, may be native withthe DNA sequence of interest, or may be derived from another source.

The “transcription initiation site” is the position surrounding thefirst nucleotide that is part of the transcribed sequence, which is alsodefined as position +1. With respect to this site all other sequences ofthe gene and its controlling regions are numbered. Downstream sequences(i.e. further protein encoding sequences in the 3′ direction) aredenominated positive, while upstream sequences (mostly of thecontrolling regions in the 5′ direction) are denominated negative.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. “Transiently transformed” refers to cells in whichtransgenes and foreign DNA have been introduced (for example, by suchmethods as Agrobacterium-mediated transformation or biolisticbombardment), but not selected for stable maintenance. “Stablytransformed” refers to cells that have been selected and regenerated ona selection media following transformnation.

“Transformed/transgenic/recombinant” refer to a host organism such as abacterium or a plant into which a heterologous nucleic acid molecule hasbeen introduced. The nucleic acid molecule can be stably integrated intothe genome of the host or the nucleic acid molecule can also be presentas an extrachromosomal molecule. Such an extrachromosomal molecule canbe auto-replicating. Transformed cells, tissues, or plants areunderstood to encompass not only the end product of a transformationprocess, but also transgenic progeny thereof A “non-transformed”,“non-transgenic”, or “non-recombinant” host refers to a wild-typeorganism, e.g., a bacterium or plant, which does not contain theheterologous nucleic acid molecule.

“Transient expression” refers to expression in cells in which a virus ora transgene is introduced by viral infection or by such methods asAgrobacterium-mediated transformation, electroporation, or biolisticbombardment, but not selected for its stable maintenance.

The term “translation leader sequence” refers to that DNA sequenceportion of a gene between the promoter and coding sequence that istranscribed into RNA and is present in the fully processed mRNA upstream(5′) of the translation start codon. The translation leader sequence mayaffect processing of the primary transcript to mRNA, mRNA stability ortranslation efficiency.

“Vector” is defined to include, inter alia, any plasmid, cosmid, phageor Agrobacterium binary vector in double or single stranded linear orcircular form which may or may not be self transmissible or mobilizable,and which can transform prokaryotic or eukarybtic host either byintegration into the cellular genome or exist extrachromosomally (e.g.autonomous replicating plasmid with an origin of replication).Specifically included are shuttle vectors by which is meant a DNAvehicle capable, naturally or by design, of replication in two differenthost organisms, which may be selected from actinomycetes and relatedspecies, bacteria and eucaryotic (e.g. higher plant, mammalian, yeast orfungal cells).

“Visible marker” refers to a gene whose expression does not confer anadvantage to a transformed cell but can be made detectable or visible.Examples of visible markers include but are not limited toβ-glucuronidase (GUS), luciferase (LUC) and green fluorescent protein(GFP).

“Wild-type” refers to the normal gene, virus, or organism found innature without any known mutation.

DETAILED DESCRIPTION OF THE INVENTION

Identification of root-specific genes, promoters and homologues.

In many instances, it is desirable to spatially regulate the expressionof a transgene so as to be expressed only in plant root tissues. Apromoter capable of directing expression in a specific or preferentialmanner can most expeditiously accomplish this spatial regulation.

The present invention provides isolated nucleic acid molecules having anucleotide sequence that directs root-specific transcription in a plant.Root-specific promoters are isolated by identifying genes that arespecifically expressed in root tissue of a target plant and subsequentlyisolating the regulatory sequences of these genes. In one method asfurther described in the examples below, a PCR-subtractive approach isused. Messenger RNA (mRNA) is isolated from maize root-tissue as well asfrom a combination of non-root tissues from maize, such as leaf, stem,and reproductive tissues. Complimentary DNA (cDNA) is made from eachmRNA population and subsequently each cDNA population is digested with arestriction enzyme. Adapter primers, short DNA sequences, are ligated tothe 5′ and 3′ ends of root-specific cDNA. The root-specific and non-rootcDNA fragment populations are then hybridized and PCR is used toselectively amplify root-specific cDNA fragments which are not presentin the non-root cDNA population and thus do not hybridize with thenon-root cDNA fragment population. The amplified cDNA fragmentsrepresent fragments of root-specific genes. Using this methodroot-specific fragments are obtained. Longer sequences, full-length ornear full-length cDNA clones of these genes can be obtained by PCRtechniques or hybridization. Using a technique known in the art as RACE(rapid amplification of cDNA ends), gene-specific primers are made tothe 5′ region of the known sequence of each root-specific fragment andPCR is performed on a root-specific cDNA library in which the 5′ end ofeach cDNA of the library has been ligated to a short nucleotide adaptersequence. PCR is used to amplify the region between the gene-specificprimer and the adapter sequence. Root-specific cDNA clones exemplifiedherein by SEQ ID NOS: 5, 7, 9, and 11, were obtained generally usingthis technique.

Promoter sequences are obtained by cloning the genomic sequences thatare homologous to the root-specific cDNA sequences. Genomic sequencesmay be obtained by hybridization methods or by using PCR methods toextend the sequence in either the 5′ or 3′ direction from the knownsequence (sometimes referred to as “genome walking”). For example, toobtain genomic sequences 5′ to the known sequence of the cDNA, primersare made to the sequence near the 5′ end of the cDNA. A genomic libraryis constructed with the 5′ end of each genomic DNA sequence ligated to ashort oligonucleotide adapter. PCR with a primer hybridizing to theadapter sequence and a 5′ primer of a root-specific cDNA sequence allowsamplification of a genomic sequence residing 5′ to the homologoussequence of the root-specific sequence. DNA sequences obtained fromgenome walking are sequenced and if additional 5′ regions are desired,the process is repeated with primers now at the 5′ end of the longestobtained clone. Genomic sequences homologous to root-specific cDNAsequences are also obtained by hybridization under high stringencyconditions. High stringency conditions select for hybridization of aprobe made from a root-specific cDNA sequence to hybridize to itshomologous sequence in the genomic DNA. The genomic DNA is comprised ina genomic DNA library of 5-20 kb maize genomic DNA sequences in a lambdaphage vector. Genomic clones that hybridize with the root-specific cDNAare isolated and sequenced.

The genomic clones may include intron sequences, not found in the mRNAor the cDNA clones. The genomic sequences may additionally comprise 5′untranslated sequences, 3′ untranslated sequences, and 5′ and 3′regulatory sequences. Promoter sequences are found within the genomicsequence 5′ to the cDNA sequence. Genomic sequences are cloned which arehomologous to the root-specific cDNA sequences. Sequences that are 5′ tothe sequence homologous to the cDNA sequence are herein referred to asthe 5′ flanking region which comprises the promoter region.

Promoter and other regulatory sequences are mapped by comparison of thegenomic sequence with the homologous root-specific cDNA sequences aswell as using sequence homology comparisons to locate the TATA box andother regulatory elements such as binding sites for known planttranscription factors (Guifoyle, T J, 1997, Genetic Engineering 19:15-47; Meisel and Lam, 1997, Genetic Engineering 19: 183-199)).Promoters exemplified herein are set forth in SEQ ID NOS: 1-4.

In one embodiment of the invention, to further delineate the sequencesrequired for root-expression as well as those regulatory sequences thatinfluence the overall level of expression, deletions of theroot-specific promoter regions are made. Deletions are made in the 5′flanking region of each root-specific clone. In most promoters 500-1,000base pairs (bp) of 5′ flanking sequence are sufficient for promoteractivity, including tissue-specific activity. Deletions of the 5′flanking region can result in promoter regions of approximately 50 bp,100 bp, 250 bp, 500 bp, 750 bp and 1,000 bp or more. These promoterdeletion sequences serve a two-fold purpose. The deletions allow thefurther mapping of regulatory sequences within the 5′ flanking sequenceof each root-specific genomic clone. Additionally, the deletions providea toolbox of promoter and regulatory sequences that vary in theirexpression levels and expression patterns thus providing additionalflexibility in choosing promoter sequences for appropriate generegulation. Exemplified herein are fully functional shorter fragments ofa promoter designated MRS1 (SEQ ID NO: 1) and a promoter designated MRS2(SEQ ID NO: 2). The fully functional shorter fragment of MRS1 comprisesnucleotides 603-1392 of SEQ ID NO: 1 and is designated MRS1 S. One fullyfunctional shorter fragment of MRS2 comprises nucleotides 921-2869 ofSEQ ID NO: 2 and is designated MRS2-M. Another fully functional shorterfragment of MRS2 comprises nucleotides 1913-2869 of SEQ ID NO: 2 and isdesignated MRS2-S.

It is also clear to one skilled in the art that mutations, insertions,deletions and/or substitutions of one or more nucleotides can beintroduced into the nucleotide sequences of SEQ ID NOS: 1-4 usingmethods known in the art. In addition, shuffling the sequences of theinvention can provide new and varied nucleotide sequences.

To test for a function of variant DNA sequences according to theinvention, such as deletion fragments of SEQ ID NOS: 1-4, the sequenceof interest is operably linked to a selectable or visible marker geneand expression of the marker gene is tested in transient expressionassays with isolated root tissue or cells or by stable transformationinto plants. It is known to the skilled artisan that DNA sequencescapable of driving expression of an associated coding sequence are builtin a modular way. Accordingly, expression levels from shorter DNAfragments may be different than the one from the longest fragment andmay be different from each other. For example, deletion of adown-regulating upstream element will lead to an increase in theexpression levels of the associated coding sequence while deletion of anup-regulating element will decrease the expression levels of theassociated coding sequence. It is also known to the skilled artisan thatdeletion of development-specific or a tissue-specific elements will leadto a temporally or spatially altered expression profile of theassociated coding sequence.

In another embodiment of the invention, DNA and genomic DNA sequenceshomologous to SEQ ID NOS: 1-4 or SEQ ID NOS: 5, 7, 9, and 11 may beisolated from other maize germplasm using either hybridization or PCRtechniques well known in the art. The isolated sequences may beidentical to SEQ ID NOS: 1-4 or SEQ ID NOS: 5, 7, 9, and 11 or they maybe substantially identical to SEQ ID NOS: 14 or SEQ ID NOS: 5, 7, 9, and11. It is not necessary for the sequences obtained from other maizegermplasm to contain identical nucleotide sequences to be functionallyidentical to the sequences disclosed herein. Some nucleotide deletions,additions, and replacements may have no impact or only a minor impact ongene expression. A preferable isolated nucleic acid molecule, accordingto the present invention, comprises a nucleotide sequence that has atleast 70% identity to any one of the nucleotide sequences set forth inSEQ ID NOS: 1-4. A more preferable isolated nucleic acid moleculecomprises a nucleotide sequence that has at least 80% identity to anyone of the nucleotide sequences set forth in SEQ ID NOS: 1-4. An evenmore preferable isolated nucleic acid molecule comprises a nucleotidesequence that has at least 90% identity to any one of the nucleotidesequences set forth in SEQ ID NOS: 1-4. An even more preferable isolatednucleic acid molecule comprises a nucleotide sequence that has at least95% identity to any one of the nucleotide sequences set forth in SEQ IDNOS: 1-4. An even more preferable isolated nucleic acid moleculecomprises a nucleotide sequence that has at least 99% identity to anyone of the nucleotide sequences set forth in SEQ ID NOS: 1-4. The mostpreferable isolated nucleic acid molecule comprises any one of thenucleotide sequences set forth is SEQ ID NOS: 1-4.

In another embodiment of the invention, cDNA and genomic DNA sequencesmay be cloned from other plants that represent homologues of theroot-specific maize genes and promoters. These homologues allow one toobtain additional root-specific promoters useful for the regulation ofmultiple genes in the root. Hybridization using the maize cDNA andgenomic sequences or portions thereof is used to screen for homologousor substantially identical sequences in other plant genomes. Thesesequences may comprise only a subset of the nucleotides or SEQ ID NOS:1-4. A preferable length of homology is 20 base pairs (bp) in length,more preferably, 50 bp in length, and most preferably at least 100 bp inlength. In one embodiment of the present invention, a hybridizationprobe is prepared from any one SEQ ID NOS: 1-4 or portions thereof orSEQ ID NOS: 5, 7, 9, or 11 or portions thereof. Hybridization of suchsequences may be carried out under high stringency conditions.Alternatively, low or moderate stringency conditions can be used toallow some mismatching in sequences so that lower degrees of similarityare detected (heterologous probing). Generally, a probe is less thanabout 1000 nucleotides in length, preferably less than 500 nucleotidesin length.

In another embodiment of the present invention, cDNA and genomicsequences are isolated by preparing primers comprising sequences withinany one of SEQ ID NOS: 1-4 or comprising primer sequences from SEQ IDNOS: 5, 7, 9, or 11. The primers may be used in a PCR reaction with cDNAor genomic DNA from a plant to obtain homologous sequences or sequenceswith substantial identity to any one of SEQ ID NOS: 1-4.

Construction of Expression Cassettes

Expression cassettes are constructed comprising the 5′ flankingsequences of the root-specific genomic clones. In one embodiment of thepresent invention, the promoter region utilized in each expressioncassette comprises the 5′ flanking region up to and including the startof translation. The start of translation is denoted by the first ATG ofthe open reading frame (ORF) found in the cDNA and the homologousgenomic sequence. Thus, the promoter region may include 5′ untranslatedleader sequence as well as the transcriptional start site, core promoterand additional regulatory elements. In another embodiment of the presentinvention, expression cassettes are constructed comprising the 5′flanking sequence of the root-specific genomic clones up to andincluding the transcriptional initiation site. The transcriptionalintiation site may be defined by the first nucleotide of the longestcDNA clone obtained. Additionally, the transcriptional intiation sitemay be further defined by use of techniques well known in the artincluding RACE PCR, RNase protection mapping and primer extensionanalysis.

The expression cassettes may further comprise a transcriptionalterminator, downstream (3′) to the promoter. A variety oftranscriptional terminators are available for use in expressioncassettes. The transcriptional terminator is responsible for thetermination of transcription beyond the transgene and correct mRNApolyadenylation of the mRNA transcript. Appropriate transcriptionalterminators are those that are known to function in plants and includethe CaMV 35S terminator, the tml terminator, the nopaline synthaseterminator and the pea rbcS E9 terminator. These can be used in bothmonocotyledons and dicotyledons. In addition, a gene's nativetranscription terminator may be used. For example, the 3′ flankingsequence comprising genomic sequence 3′ to the region homologous to aroot-specific cDNA clone may be used.

In a preferred embodiment of the present invention a heterologous codingsequence, for example, an insecticidal coding sequence, a visible markercoding sequence, or a selectable marker coding sequence, is clonedbetween a promoter of the: invention and transcriptional terminatorwhereby the heterologous coding sequence is operatively linked to thepromoter and the transcriptional terminator is operatively linked to theheterologous coding sequence. Examples of visible markers useful for thepresent invention include, but are not limited to, β-glucuronidase(GUS), Chloramphenicol Acetyl Transferase (CAT), Luciferase (LUC) andproteins with fluorescent properties, such as Green Fluorescent Protein(GFP) from Aequora victoria. In principle, many more proteins aresuitable for this purpose, provided the protein does not interfere withessential plant functions. Further examples of heterologous codingsequences useful for the present invention include, but are not limitedto, antibiotic resistance, virus resistance, insect resistance, diseaseresistance, or resistance to other pests, herbicide tolerance, improvednutritional value, improved performance in an industrial process oraltered reproductive capability. In a preferred embodiment of thepresent invention, a gene encoding for resistance to insects that feedon the roots of the plant is cloned between the promoter and terminator.In another embodiment of the present invention it sequence encoding afunctional RNA such as antisense RNA, a sense RNA for sense-suppression,or a double stranded RNA may also be cloned between the promoter andtranscriptional terminator.

Numerous sequences have been found to enhance gene expression fromwithin the transcriptional unit and these sequences can be used inconjunction with the promoters of this invention to increase theirexpression in transgenic plants. Various intron sequences have beenshown to enhance expression, particularly in monocotyledonous cells. Forexample, the introns of the maize AdhI gene have been found tosignificantly enhance the expression of the wild-type gene under itscognate promoter when introduced into maize cells. Intron 1 was found tobe particularly effective and enhanced expression in fusion constructswith the chloramphenicol acetyltransferase gene (Callis et al., GenesDevelop. 1: 1183-1200 (1987)). In the same experimental system, theintron from the maize bronze1 gene had a similar effect in enhancingexpression. Intron sequences have been routinely incorporated into planttransformation vectors, typically within the non-translated leader. Anumber of non-translated leader sequences derived from viruses are alsoknown to enhance expression, and these are particularly effective indicotyledonous cells. Specifically, leader sequences from Tobacco MosaicVirus (TMV, the “W-sequence”), Maize Chlorotic Mottle Virus (MCMV), andAlfalfa Mosaic Virus (AMV) have been shown to be effective in enhancingexpression (e.g. Gallie et al. Nucl. Acids. Res. 15: 8693-8711 (1987);Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)). Other leadersequences known in the art include but are not limited to: picornavirusleaders, for example, EMCV leader (Encephalomyocarditis 5′ noncodingregion) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. PNAS USA86:6126-6130 (1989)); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al., 1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9-20); human immunoglobulin heavy-chainbinding protein (BiP) leader, (Macejak, D. G., and Sarnow, P., Nature353: 90-94 (1991); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L.,Nature 325:622-625 (1987); tobacco mosaic virus leader (TMV), (Gallie,D. R. et al., Molecular Biology of RNA, pages 237-256 (1989); and MaizeChlorotic Mottle Virus leader (MCMV) (Lommel, S. A. et al., Virology81:382-385 (1991). See also, Della-Cioppa et al., Plant Physiology84:965-968 (1987).

Plant Transformation Methods Useful for the Invention

Numerous transformation vectors available for plant transformation areknown to those of ordinary skill in the plant transformation art, andthe nucleic acid molecules of the invention can be used in conjunctionwith any such vectors. The selection of vector will depend upon thepreferred transformation technique and the target plant species fortransformation. For certain target species, different antibiotic orherbicide selection markers may be preferred. Selection markers usedroutinely in transformation include the nptII gene, which confersresistance to kanamycin and related antibiotics (Messing & Vierra. Gene19: 259-268 (1982); Bevan et al., Nature304:184-187 (1983)), the bargene, which confers resistance to the herbicide phosphinothricin (Whiteet al., Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl.Genet 79: 625-631 (1990)), the hph gene, which confers resistance to theantibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4:2929-2931), and the dhfr gene, which confers resistance to methatrexate(Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)), the EPSPS gene, whichconfers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and5,188,642), and the mannose-6-phosphate isomerase gene, which providesthe ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and5,994,629).

Vectors Suitable for Agrobacterium Transformation

Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA border sequenceand include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)).Below, the construction of two typical vectors suitable forAgrobacterium transformation is described.

pCIB200 and pCIB2001:

The binary vectors pCIB200 and pCIB2001 are used for the construction ofrecombinant vectors for use with Agrobacterium and are constructed inthe following manner. pTJS75kan is created by NarI digestion of pTJS75(Schmidhauser & Helinski, J. Bacteriol. 164: 446-455 (1985)) allowingexcision of the tetracycline-resistance gene, followed by insertion ofan AccI fragment from pUC4K carrying an NPTII (Messing & Vierra, Gene19: 259-268 (1982): Bevan et al., Nature 304: 184-187 (1983): McBride etal., Plant Molecular Biology 14: 266-276 (1990)). XhoI linkers areligated to the EcoRV fragment of PCIB7 which contains the left and rightT-DNA borders, a plant selectable nos/nptII chimeric gene and the pUCpolylinker (Rothstein et al., Gene 53: 153-161 (1987)), and theXhol-digested fragment are cloned into SalI-digested pTJS75kan to createpCIB200 (see also EP 0 332 104, example 19). pCIB200 contains thefollowing unique polylinker restriction sites: EcoRI, SstI, KpnI, BglII,XbaI, and SalI. pCIB2001 is a derivative of pCIB200 created by theinsertion into the polylinker of additional restriction sites. Uniquerestriction sites in the polylinker of pCIB2001 are EcoRI, SstI, KpnI,BglII, XbaI, SalI, MluI, BelI, AvrII, ApaI, HpaI, and Stul. pCIB2001, inaddition to containing these unique restriction sites also has plant andbacterial kanamycin selection, left and right T-DNA borders forAgrobacterium-mediated transformation, the RK2-derived trfA function formobilization between E. coli and other hosts, and the OrlT and OriVfunctions also from RK2. The pCIB2001 polylinker is suitable for thecloning of plant expression cassettes containing their own regulatorysignals.

pCIB10 and Hygromycin Selection Derivatives thereof:

The binary vector pCIB10 (Rothstein et al. (Gene 53: 153-161 (1987))contains a gene encoding kanamycin resistance for selection in plantsand T-DNA right and left border sequences and incorporates sequencesfrom the wide host-range plasmid pRK252 allowing it to replicate in bothE. coli and Agrobacterium. Various derivatives of pCIB10 thatincorporate the gene for hygromycin B phosphotransferase are describedby Gritz et al. (Gene 25: 179-188 (1983)). These derivatives enableselection of transgenic plant cells on hygromycin only (pCIB743), orhygromycin and kanamycin (pCIB715, pCIB717).

Vectors Suitable for Non-Agrobacterium Transformation

Transformation without the use of Agrobacterium tumefaciens circumventsthe requirement for T-DNA sequences in the chosen transformation vectorand consequently vectors lacking these sequences can be utilized inaddition to vectors such as the ones described above which contain T-DNAsequences. Transformation techniques that do not rely on Agrobacteriuminclude transformation via particle bombardment, protoplast uptake (e.g.PEG and electroporation) and microinjection. The choice of vectordepends largely on the preferred selection for the species beingtransformed. Below, the construction of typical vectors suitable fornon-Agrobacterium transformation is described.

pCIB3064:

pCIB3064 is a pUC-derived vector suitable for direct gene transfertechniques in combination with selection by the herbicide Basta (orphosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoterin operational fusion to the E. coli GUS gene and the CaMV 35Stranscriptional terminator and is described in the PCT publishedapplication WO 93/07278. The 35S promoter of this vector contains twoATG sequences 5′ of the start site. These sites are mutated usingstandard PCR techniques in such a way as to remove the ATGs and generatethe restriction sites SspI and PvuII. The new restriction sites are 96and 37 bp away from the unique SalI site and 101 and 42 bp away from theactual start site. The resultant derivative of pCIB246 is designatedpCIB3025. The GUS gene is then excised from pCIB3025 by digestion withSalI and SacI, the termini rendered blunt and re-ligated to generateplasmid pCIB3060. The plasmid pJIT82 is obtained from the John InnesCentre, Norwich and the 400 bp Smal fragment containing the bar genefrom Streptomyces viridochromogenes is excised and inserted into theHpaI site of pCIB3060 (Thompson et al. EMBO J 6: 2519-2523 (1987)). Thisgenerated pCIB3064, which comprises the bar gene under the control ofthe CaMV 35S promoter and terminator for herbicide selection, a gene forampicillin resistance (for selection in E. coli) and a polylinker withthe unique sites SphI, PstI, HindIII, and BamHI. This vector is suitablefor the cloning of plant expression cassettes containing their ownregulatory signals.

pSOG19 and pSOG35:

pSOG35 is a transformation vector that utilizes the E. coli genedihydrofolate reductase (DFR) as a selectable marker conferringresistance to methotrexate. PCR is used to amplify the 35S promoter(−800 bp), intron 6 from the maize Adhl gene (−550 bp) and 18 bp of theGUS untranslated leader sequence from pSOG10. A 250-bp fragment encodingthe E. coli dihydrofolate reductase type II gene is also amplified byPCR and these two PCR fragments are assembled with a SacI-PstI fragmentfrom pB1221 (Clontech) which comprises the pUC19 vector backbone and thenopaline synthase terminator. Assembly of these fragments generatespSOG19 that contains the 35S promoter in fusion with the intron 6sequence, the GUS leader, the DHFR geno and the nopaline synthaseterminator. Replacement of the GUS leader in pSOG19 with the leadersequence from Maize Chlorotic Mottle Virus (MCMV) generates the vectorpSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistanceand have HindIII, SphI, PstI and EcoRI sites available for the cloningof foreign substances.

Transformation Methods Useful for the Present Invention

Once a nucleic acid molecule of the invention has been cloned into anexpression cassette, it is transformed into a plant cell. The receptorand target expression cassettes of the present invention can beintroduced into the plant cell in a number of art-recognized ways.Methods for regeneration of plants are also well known in the art. Forexample, Ti plasmid vectors have been utilized for the delivery offoreign DNA, as well as direct DNA uptake, liposomes, electroporation,microinjection, and microprojectiles. In addition, bacteria from thegenus Agrobacterium can be utilized to transform plant cells. Below aredescriptions of representative techniques for transforming bothdicotyledonous and monocotyledonous plants, as well as a representativeplastid transformation technique.

Plants transformed in accordance with the present invention may bemonocots or dicots and include, but are not limited to, maize, wheat,barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage,cauliflower, broccoli, turnip, radish, spinach, asparagus, onion,garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear,quince, melon, plum, cherry, peach, nectarine, apricot, strawberry,grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana,soybean, tomato, sorghum, sugarcane, sugarbeet, sunflower, rapeseed,clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant,cucumber, Arabidopsis thaliana, and woody plants such as coniferous anddeciduous trees, especially maize, wheat, or sugar beet.

Once an expression cassette is transformed into a particular plantspecies, the expression cassette may be propagated in that species ormoved into other varieties of the same species, particularly includingcommercial varieties, using traditional breeding techniques.

Transformation of Dicotyledons

Transformation techniques for dicotyledons are well known in the art andinclude both Agrobacterium-based and non-Agrobacterium based techniques.Non-Agrobacterium techniques involve the uptake of exogenous geneticmaterial directly by protoplasts or cells. This can be accomplished byparticle bombardment-mediated delivery, microinjection, or PEG orelectroporation mediated uptake. Examples of these techniques aredescribed by Paszkowski et al., EMBO J 3: 2717-2722 (1984), Potrykus etal., Mol. Gen. Genet. 199: 169-177 (1985), Reich et al., Biotechnology4: 1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987). In eachcase the transformed cells are regenerated to whole plants usingstandard techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique fortransformation of dicotyledons because of its high efficiency oftransformation and its broad utility with many different species.Agrobacterium transformation typically involves the transfer of thebinary vector carrying the foreign DNA of interest (e.g. pCIB200 orpCIB2001) to an appropriate Agrobacterium strain which may depend of thecomplement of vir genes carried by the host Agrobacterium strain eitheron a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 forpCIB200 and pCMB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). Thetransfer of the recombinant binary vector to Agrobacterium isaccomplished by a tri-parental mating procedure using E. coli carryingthe recombinant binary vector, a helper E. coli strain which carries aplasmid such as pRK2013 and which is able to mobilize the recombinantbinary vector to the target Agrobacterium strain. Alternatively, therecombinant binary vector can be transferred to Agrobacterium by DNAtransformation (Höfgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).

Transformation of the target plant species by recombinant Agrobacteriumusually involves co-cultivation of the Agrobacterium with explants fromthe plant and follows protocols well known in the art. Transformedtissue is regenerated on selectable medium carrying the antibiotic orherbicide resistance marker present between the binary plasmid T-DNAborders.

Another approach to transforming plant cells with a gene involvespropelling inert or biologically active particles at plant tissues andcells. This technique is disclosed in U.S. Pat. Nos. 4,945,050,5,036,006, and 5,100,792 all to Sanford et al. Generally, this procedureinvolves propelling inert or biologically active particles at the cellsunder conditions effective to penetrate the outer surface of the celland afford incorporation within the interior thereof. When inertparticles are utilized, the vector can be introduced into the cell bycoating the particles with the vector containing the desired gene.Alternatively, the vector can surround the target cell so that thevector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried yeast cells, dried bacteriumor a bacteriophage, each containing DNA sought to be introduced) canalso be propelled into plant cell tissue.

Transformation of Monocotyledons

Transformation of most monocotyledon species has now also becomeroutine. Preferred techniques include direct gene transfer intoprotoplasts using PEG or electroporation techniques, and particlebombardment into callus tissue. Transformations can be undertaken with asingle DNA species or multiple DNA species (i.e. co-transformation) andboth these techniques are suitable for use with this invention.Co-transformation may have the advantage of avoiding complete vectorconstruction and of generating transgenic plants with unlinked loci forthe gene of interest and the selectable marker, enabling the removal ofthe selectable marker in subsequent generations, should this be regardeddesirable. However, a disadvantage of the use of co-transformation isthe less than 100% frequency with which separate DNA species areintegrated into the genome (Schocher et al. Biotechnology 4: 1093-1096(1986)).

Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describetechniques for the preparation of callus and protoplasts from an eliteinbred line of maize, transformation of protoplasts using PEG orelectroporation, and the regeneration of maize plants from transformedprotoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Frommet al. (Biotechnology 8: 833-839 (1990)) have published techniques fortransformation of A188-derived maize line using particle bombardment.Furthermore, WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200(1993)) describe techniques for the transformation of elite inbred linesof maize by particle bombardment. This technique utilizes immature maizeembryos of 1.5-2.5 mm length excised from a maize ear 14-15 days afterpollination and a PDS-1000He Biolistics device for bombardment.

Transformation of rice can also be undertaken by direct gene transfertechniques utilizing protoplasts or particle bombardment.Protoplast-mediated transformation has been described for Japonica-typesand Indica-types (Zhang et al. Plant Cell Rep 7: 379-384 (1988);Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology8: 736-740 (1990)). Both types are also routinely transformable usingparticle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).Furthermore, WO 93/21335 describes techniques for the transformation ofrice via electroporation.

Patent Application EP 0 332 581 describes techniques for the generation,transformation and regeneration of Pooideae protoplasts. Thesetechniques allow the transformation of Dactylis and wheat. Furthermore,wheat transformation has been described by Vasil et al. (Biotechnology10: 667-674 (1992)) using particle bombardment into cells of type Clong-term regenerable callus, and also by Vasil et al. (Biotechnology11: 1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084(1993)) using particle bombardment of immature embryos and immatureembryo-derived callus. A preferred technique for wheat transformation,however, involves the transformation of wheat by particle bombardment ofimmature embryos and includes either a high sucrose or a high maltosestep prior to gene delivery. Prior to bombardment, embryos that are0.75-1 mm in length are plated onto MS medium with 3% sucrose (Murashiga& Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/l 2,4-D forinduction of somatic embryos, which is allowed to proceed in the dark.On the chosen day of bombardment, embryos are removed from the inductionmedium and placed onto the osmoticum (i.e. induction medium with sucroseor maltose added at the desired concentration, typically 15%). Theembryos are allowed to plasmolyze for 2-3 h and are then bombarded.Twenty embryos per target plate is typical, although not critical. Anappropriate gene-carrying plasmid (such as pCIB3064 or pSG35) isprecipitated onto micrometer size gold particles using standardprocedures. Each plate of embryos is shot with the DuPont Biolistics®helium device using a burst pressure of ˜1000 psi using a standard 80mesh screen. After bombardment, the embryos are placed back into thedark to recover for about 24 h (still on osmoticum). After 24 hrs, theembryos are removed from the osmoticum and placed back onto inductionmedium where they stay for about a month before regeneration.Approximately one month later the embryo explants with developingembryogenic callus are transferred to regeneration medium (MS+1 mg/literNAA, 5 mg/liter GA), further containing the appropriate selection agent(10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in thecase of pSOG35). After approximately one month, developed shoots aretransferred to larger sterile containers known as “GA7s” which containhalf-strength MS, 2% sucrose, and the same concentration of selectionagent.

Transformation of monocotyledons using Agrobacterium has also beendescribed in WO 94/00977 and U.S. Pat. No. 5,591,616, both of which areincorporated herein by reference. A preferred method of maizetransformation is described in Negrotto et al., (Plant Cell Reports 19:798-803 (2000)), incorporated herein by reference.

Analysis of Promoter Activity

Several methods are available to assess promoter activity. Expressioncassettes are constructed with a visible marker, as described above.Transient transformation methods: are used to assess promoter activity.Using transformnation methods such as microprojectile bombardment,Agrobacterium transformation or protoplast transformation, expressioncassettes are delivered to plant cells or tissues. Reporter geneactivity, such as β-glucuronidase activity, luciferase activity or GFPfluorescence is monitored after transformation over time, for example 2hours, 5 hours, 8 hours, 16 hours, 24 hours, 36 hours, 48 hours and 72hours after DNA delivery using methods well known in the art. Reportergene activity may be monitored by enzymatic activity, by staining cellsor tissue with substrate for the enzyme encoded by the reporter gene orby direct visualization under an appropriate wavelength of light.Full-length promoter sequences, deletions and mutations of the promotersequence may be assayed and their expression levels compared.Additionally, RNA levels may be measured using methods well known in theart such as Northern blotting, competitive reverse transcriptase PCR andRNAse protection assays. These assays measure the level of expression ofa promoter by measuring the ‘steady state’ concentration of a standardtranscribed reporter mRNA. This measurement is indirect since theconcentration of the reporter mRNA is dependent not only on itssynthesis rate, but also on the rate with which the mRNA is degraded.Therefore the steady state level is the product of synthesis rates anddegradation rates. The rate of degradation can however be considered toproceed at a fixed rate when the transcribed sequences are identical,and thus this value can serve as a measure or synthesis rates.

Further confirmation of promoter activity is obtained by stabletransformation of the promoter in an expression cassette comprising avisible marker or gene of interest into a plant as described above.Using the various methods described above such as enzymatic activityassays, RNA analysis and protein assays as described supra, promoteractivity is monitored over development, and additionally by monitoringexpression in different tissues in the primary transformants and throughsubsequent generations of transgenic plants.

EXAMPLES

The invention will be further described by reference to the followingdetailed examples. These examples are provided for purposes ofillustration only, and are not intended to be limiting unless otherwisespecified. Standard recombinant DNA and molecular cloning techniquesused here are well known in the art and are described by Ausubel (ed.),Current Protocols in Molecular Biology, John Wiley and Sons, Inc.(1994); J. Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3dEd., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press(2001); and by T. J. Silhavy, M. L. Berman, and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1984).

Example 1 Construction of Maize Root Forward and Reverse Subtracted cDNALibraries

As a first step towards the identification of maize root-specific cDNAclones, subtracted cDNA libraries were constructed using Clontech'sPCR-Select cDNA Subtraction Kit (Clontech Cat. No. K1804-1). Twodifferent subtracted cDNA libraries were constructed; 1) aforward-subtracted library enriched for differentially expressedtranscripts that are specific to the root, and 2) a reverse-subtractedlibrary enriched for differentially expressed transcripts that arespecific to all aboveground tissues exclusive of roots. Total RNA wasisolated from maize line CG00526 including tassel, silks, seed, stem,leaf, and roots using tissue samples pooled from mature and severaldevelopmental stages for each organ. For the root, crown roots, primaryroots and lateral roots were sampled from approximately five evenlydivided developmental stages starting two weeks post germination andending at maturity. Poly A mRNA was isolated from each of the total RNAprops using procedures outlined in the PolyATtract mRNA isolationSystems kit (Promega Cat. No. Z3790). cDNAs were synthesized from polyAmRNA from each organ, digested with Rsa I, ligated to adaptors, anddivided into “tester cDNA”, and “driver cDNA” populations. For theforward-subtracted library the “tester cDNA” was represented by the rootcDNA and the “driver cDNA” was composed of equal quantities of tassel,silk, seed, stem, and leaf cDNAs. For the reverse-subtracted library the“tester” and “driver” cDNA populations were the reverse of those used inthe forward-subtracted library.

The cDNA subtraction and PCR amplification for each of the libraries wascarried out as described in the user's manual of the Clontech PCR-SelectcDNA Subtraction Kit. The PCR products of the root-specificforward-subtracted library were cloned into both the TA-cloning vectorpCR2.1 (Invitrogen Cat. No. K20000-01) and into pBSK (Stratagene Cat.No. 212206). cDNAs ligated into pBSK were transformed into E. colistrain DH10B cells (Life Technologies Cat. No.18290-015) byelectroporation. Transformation reactions were plated onto Q-trays(Genetix) containing media enabling blue/white colony selection. Whitecolonies on Q-trays were robotically picked (Q-bot) into 384-well platescontaining antibiotic selection media and cryoprotectant. Colonies in384-well plates were grown up and frozen-off at −80° C. Approximately16,000 colonies were picked. Each of the approximately 16,000 griddedbacterial colonies representing the maize root subtracted library wasrobotically arrayed as duplicate spots on replicate 23 cm² nylonmembranes using a Q-bot (Genetix Limited). Colonies were grown up onmembranes overlaid onto selective agar trays and subsequent in situbacterial colony lysis and processing of membrane for hybridization wereperformed according to Nizetic et al. (Nucleic Acids Res. 19(1): 182,(1991)).

Example 2 Screening of Maize Root Subtracted cDNA Library, Sequencing ofClones and Confirmation of Root Specificity by Northern Blot Analysis

Differential hybridization screening of the arrayed library with³²P-labeled probes were performed in order to identify clones whoseexpression was specific to the root. This was accomplished byhybridizing one of the two replicate arrays of the library with aforward-subtracted probe made from the same subtracted cDNA that wasused to construct the forward-subtracted library; and hybridizing thesecond replica array with a reverse-subtracted probe using the samesubtracted cDNA used to construct the reverse-subtracted library. Probeproduction and hybridizations were performed according to the usersmanual of the PCR-Select Differential Screening Kit (Clontech Cat. No.K1808-1, -D). Nylon membranes were exposed to both film (Kodak Biomax)and phosphoimager screens and densitometry values were assigned to eachof the spots.

Using visual inspection of autoradiographs and densitometry data, 300clones were picked for sequencing. Clones were picked on their abilityto satisfy three criteria; 1) No hybridization to the reverse-subtractedprobe, 2) Strong hybridization to the forward-subtracted probe, and 3)Consistent hybridization intensity between duplicate spots on the samefilter. The 300 sequences were arranged into 70 unique contigs using thePhred/Phrap program (Codon Code Corp.) for sequence analysis and contigassembly. Sequence identities were searched using the Blast program andclones giving the same blast ID were eliminated. The resulting 36 uniqueclones were then evaluated for root-specific expression by northern blotanalysis.

Gel blots containing 18 μg of total RNA (isolated from tissues atseveral developmental stages for each sample) from each of six differentorgans, tassel, silk, seed, leaf, stem, and root were hybridized with³²P-labeled probes made from each of the 36 cDNAs. These analysesconfirmed that 15 clones (2A12, 10B21, 4J8, 16M14, 29D21, 7B21, 14H6,2D14, 16H17, 36E7, 4H19, 2P9, 6G15, 22P8, and SM13) had root-specificexpression.

The 15 cDNA clones from above were then subjected to reverse northernblot analysis to evaluate whether any of the previously observedroot-specific expression might be due to expression that is confined toor predominant in the root tip. Five duplicate gel blots of the 15root-specific cDNA clones were prepared and each was hybridized with afirst-strand ³²P-labeled cDNA probe prepared from polyA⁺ RNA isolatedfrom either whole roots, root-tips, roots without tips, crown root tips,or crown roots without tips. The intensity of the ³²P-labeled band onthe autoradiograph was characterized as high (+++), medium (++), low(+), or absent (−). The results shown in Table 1 indicate thatexpression of all 15 clones was not confined to the root tip and thatsubstantial expression was found throughout the root.

TABLE 1 Results of Northern and Reverse Northern Analysis. RelativeRelative Levels of mRNA in Reverse Northern Levels of Roots Crown mRNAin Whole w/o Crown roots w/o Clone Northern roots Root tips root tipsroot tips root tips 2A12 +++ + + + − + 2D14 ++ + +++ ++ +++ +++ 2P9++ + + + − + 4H19 +++ +++ + +++ − ++ 4J8 +++ + + + + + 6G15 ++ + + + + +7B12 +++ +++ + +++ − ++ 10B21 ++ ++ +++ ++ +++ +++ 14H6 ++ + + + + +16H17 +++ +++ + +++ + +++ 16M14 + +++ +++ +++ ++ ++ 22P8 +++ +++ ++ +++− +++ 29D21 + ++ − + − 36E7 +++ +++ + +++ − +++ SM13 +++ +++ +++ +++ ++−

Example 3 Obtaining full-length cDNA Clones for Selected Root-specificcDNAs

Using the above northern and reverse northern expression data, fourclones, 22P8, 10B21, 2D14, 4H19 were chosen to obtain full-length cDNAs.All four of the original cDNAs for these clones represent 3′ fragmentseach containing poly A tails. 5′ RACE PCR using Clontech's Marathon cDNAAmplification Kit (Cat. No. K1802-1) was used to extend the partial cDNAclones. Because of the high G+C content of maize DNA, incremental RACEextensions utilizing the following RACE gene-specific primers (SEQ IDNOS: 13-26) and the adapter primer (SEQ ID NO: 27) were used to obtainthe full-length cDNA products:

Gene Specific Primers used for Each Designated Clone

Clone Primer Sequence SEQ ID NO 22P8 5′-CACACTAGCGCACAGAGATCAGAG-3′ (SEQID NO: 13) 5′-GCACCAACACAAGCACAACAGAAC-3′ (SEQ ID NO: 14)5′-CAGGGTACATCTTGCCGCACTTGC-3′ (SEQ ID NO: 15)5′-TGATCCGCAGTTGCAGCTTGATCC-3′ (SEQ ID NO: 16) 10B215′-CATGCTCGCGGCTAGCTTAGAGG-3′ (SEQ ID NO: 17)5′-ACAGTCTTGCCGCAGTGGTTGAG-3′ (SEQ ID NO: 18)5′-TGAGGCTGAGGTCGACGGGCAGG-3′ (SEQ ID NO: 19)5′-CGAGGTCGACGAGTCCCTTCAGC-3′ (SEQ ID NO: 20) 2D145′-TTAGCACCGGCGTAGAAGTGATCG-3′ (SEQ ID NO: 21)5′-GCATGGAATGGAAGGGAGGCAGC-3′ (SEQ ID NO: 22) 4H195′-ATACAACGCAAGGTTCGCTCACTG-3′ (SEQ ID NO: 23)5′-CATGACCGCTAAGGATCAGGAGAC-3′ (SEQ ID NO: 24)5′-TCTTTCTTGTGCACCGCCGAAGC-3′ (SEQ ID NO: 25)5′-TGCACTCACACCGCCGATGATGG-3′ (SEQ ID NO: 26) Adaptor5′-CCATCCTAATACGACTCACTATAGGGC-3′ (SEQ ID NO: 27)

The results of the RACE extensions of the cDNA clones and the primersutilized for this process are shown in Table 2. A full-length cDNAcandidate (SEQ ID Nos: 5, 7, 9, and 11) was obtained for each of thefour clones. The full-length or near full-length cDNA clone obtained byRACE PCR allowed for the prediction of the translation initiating ATGfor each of the cDNA clones.

TABLE 2 Results of cDNA RACE Extension. cDNA size Original Predictedsize 5′ RACE obtained cDNA of full-length primer by RACE cDNA obtainedClone size (bp) cDNA (bp) name (bp) by RACE 22P8 345 650-700 SEQ ID NO:13 700 SEQ ID NO: 5 SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 16 10B21 406700-800 SEQ ID NO: 17 766 SEQ ID NO: 7 SEQ ID NO: 18 SEQ ID NO: 19 SEQID NO: 20 2D14 306 700-800 SEQ ID NO: 21 730 SEQ ID NO: 9 SEQ ID NO: 224H19 311 700-850 SEQ ID NO: 23 660 SEQ ID NO: 11 SEQ ID NO: 24 SEQ IDNO: 25 SEQ ID NO: 26

Example 4 Isolation of Promoter/genomic Clones Corresponding to theRoot-specific cDNAs

Promoter/genomic clones corresponding to cDNAs 22P8, 10B21, 2D14, 4H19(SEQ ID NOS: 5, 7, 9, and 11, respectively) were isolated via PCRutilizing either maize “Genome Walker” or λ EMBL3 genomic libraries astemplate. “Genome walker” adaptor-ligated maize genomic libraries wereconstructed using DNA from maize line CGC000526 and Clontech's UniversalGenome Walker kit (Clontech Cat. No. K1807-1). Five different librarieswere constructed, each comprised of genomic DNA fragments generated bydigestion with one of the following blunt end restriction enzymes: DraI,EcoRV, HincII, SspI, or StuI. Genome Walker libraries were screenedutilizing the following gene-specific primers (SEQ ID NOS: 28-34) andthe adapter primer (SEQ ID NO: 35) which was supplied in the kit:

Gene Specific Primers Used for Each Designated Clone

Clone Primer Sequence SEQ ID NO. 22P85′-TCCTCGAGCTCTTTCGTTTGCTTTGGAAAC-3′ (SEQ ID NO: 28) 10B215′-ACACCACCAGGTTCACGGCGAGGAACAG-3′ (SEQ ID NO: 29)5′-GAGGCCTTGCCTGCCATTGCTGCAGAGT-3′ (SEQ ID NO: 30) 2D145′-AGCAGTTGGACGTGCAGGCGTTGGCTAC-3′ (SEQ ID NO: 31)5′-AGGTTCACGGCCAGGAACAGCGCGAAT-3′ (SEQ ID NO: 32) 4H195′-TCTTTCTTGTGCACCGCCGAAGC-3′ (SEQ ID NO: 33)5′-TGCACTCACACCGCCGATGATGG-3′ (SEQ ID NO: 34) Adaptor5′-GTAATACGACTCACTATAGGGC-3′ (SEQ ID NO: 35)

PCR conditions were those suggested in the Clontech users manual. PCRreactions were fractionated on agarose gels, and the resulting productband was excised, cloned in a Topo vector and sequenced to verify thecorrect product. Promoter clones corresponding to cDNAs 22P8, 10B21,2D14, 4H19 (SEQ ID NOS: 5, 7, 9, and 11, respectively) were cloned fromthese libraries.

The λ EMBL3 library was custom made by Clontech (Clontech Cat. No.cs1012j) using genomic DNA from maize line CG000526. The 1.4 kb promoterclone corresponding to cDNA 22P8 (SEQ ID NO: 5) was cloned from thislibrary. The library was PCR screened using 22P8GSP7,

5′-ACTATAGGGCACGCGTGGT-3′ (SEQ ID NO: 36)in conjunction with the following 5′ and 3′λ arm primers supplied byClontech (Cat. No. 9104-1):

(SEQ ID NO: 37) 5′-CTGCTTCTCATAGAGTCTTGCAGACAAACTGCGCAAC-3′ (SEQ ID NO:38) 5′-TGAACACTCGTCCGAGAATAACGAGTGGATCTGGGTC-3′PCR was done using conditions suggested in the user manual supplied withthe primers. The 22P8 promoter clone was a product of the 3′λ primer and22P8GSP7 primer.

Example 5 Construction of Root-specific Expression Cassettes

Entry Vectors

The first step in construction of expression cassettes was the cloningor the promoters into an entry vector. PCR primers were designed toamplify each promoter and to place attB1 and attB2 sites on the 5′ and3′ ends, respectively. The promoter corresponding to the cDNA 22P8 wasdesignated as Maize Root-Specific 1 or MRS1. Two different promoterconstructs were made from MRS1; the full-length MRS1 comprisingnucleotides 1 to 1391 of SEQ ID NO: 1, designated MRS1L, and a secondshorter fragment of MRS1 comprising nucleotides 601 to 1391 of SEQ IDNO: 1, designated MRS1S. MRS1L was PCR amplified out of Genome Walkerlibrary EcoRV with primers 22P8DS 15′-TTAAGAACATGACGGATGAAGAATCACT-3′(SEQ ID NO: 39) and 22P8GSP6 5′-TGATCCGCAGTTGCAGCTTGATCC-3′ (SEQ ID NO:40) followed by 22P8DS1 (SEQ ID NO: 39) and the nested primer 22P8GSP7(SEQ ID NO: 36). Att sites were added to MRS1L by PCR amplifying theproduct of the nested reaction with primers consisting of ½ att and ½gene-specific sequence, 22P8DS5 5′-ACAAAAAAGCAGGCTGAACATGACGGATGA-3′(SEQ ID NO: 41) and 22P8DS9 5′-ACAAGAAAGCTGGGTCCTCGAGCTCTTTCG-3′ (SEQ IDNO: 42). The full attB1 and attB2 sites were added by using primer attB15′-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3′ (SEQ ID NO: 43) and attB25′-GGGGACCACTTTGTACAAGAAAGCTGGGT-3′ (SEQ ID NO: 44). MRS1S (nucleotides601-1391 of SEQ ID NO: 1) was PCR amplified out of Genome Walker libraryEcoRV with primers 22P8DS1 (SEQ ID NO: 39) and 22P8GSP6 (SEQ ID NO: 40)followed by the nested primers 22P8DS3 5′-GCGTAGTTGAAGCTTACAAAGTTGCAT-3′(SEQ ID NO: 45) and 22P8GSP7 (SEQ ID NO: 36). Att sites were added toMRS1S by PCR amplifying the product of the nested reaction with primersconsisting of ½ att and ½ gene-specific sequence, 22P8DS75′-ACAAAAAAGCAGGCTGTAGTTGAAGCTTAC-3′ (SEQ ID NO: 46) and 22P8DS9 (SEQ IDNO: 42). The full attB1 and attB2 sites were added by using primersattB1 (SEQ ID NO: 43) and attB2 (SEQ ID NO: 44).

The promoter corresponding to the cDNA 10B21 was designated as MRS2L(SEQ ID NO: 2). MRS2L was amplified out of Genome Walker library EcoRVby PCR amplification with primers 10B21DS1,5′-GACGGCCCGGGCTGGTAAATTGACTT-3′ (SEQ ID NO: 47) and 10B21GSP1,5′-CATGCTCGCGGCTAGCTTAGAGG-3′ (SEQ ID NO: 48) followed by the nestedprimers 10B21DS3, 5′-TGGTAAATTGACTTTGCCTAGTGTTGGA-3′ (SEQ ID NO: 49) and10B21GSP2, 5′-ACAGTCTTGCCGCAGTGGTTGAG-3′ (SEQ ID NO: 50). Att sites wereadded to MRS2 by PCR amplifying the product of the nested reaction withprimers consisting of ½ att and ½ gene-specific sequence, 10B21DS5,5′-ACAAAAAAGCAGGCTTGACTTTGCCTAGTG-3′ (SEQ ID NO: 51) and 10B21DS7,5′-ACAAGAAAGCTGGGTTGCTGCAGAGTACAG-3′ (SEQ ID NO: 52). The full attB1 andattB2 sites were added by using primers attB1 (SEQ ID NO: 43) and attB2(SEQ ID NO: 44). Two shorter fragments of MRS2L were made. One fragmentof MRS2L comprising nucleotides 921 to 2869 of SEQ ID NO: 2, designatedMRS2M, and a second fragment of MRS2L comprising nucleotides 1913 to2869 of SEQ ID NO: 2, designated MRS2S. Fragments of the MRS2L promoterwere generated with PCR using the MRS2L promoter (SEQ ID NO: 2) astemplate and gene specific primers (SEQ ID NOS: 64-67). The sequenceCACC was added to the 5′ end of each forward primer to allow directionalcloning into the pENTR-D/TOPO plasmid (Invitrogen). This directionalcloning produced an entry vector comprising either the MRS2M fragment orthe MRS2S fragment. MRS2-specific primers used to make the MRS2 promoterfragments were as follows:

Primer Primer Sequence SEQ ID NO MRS2 5′-GCTGCAGAGTACAGAAAGCA-3′ SEQ IDNO: 64 Rev MRS2L 5′-CACCGGCTTGACTTTGCCTAGTGT-3′ SEQ ID NO: 65 Fwd MRS2M5′-CACCTGCGCGTGTTCGTAGAGTTG-3′ SEQ ID NO: 66 Fwd MRS2S5′-CACCTGAACTTGTGCACGTCATTT-3′ SEQ ID NO: 67 FwdCombinations of PCR primers used to generate the MRS2 promoter fragmentswere as follows:

Location on Primer Pair Product Size SEQ ID NO: 2 MRS2M Fwd + MRS2 RevMRS2M 1949  921 to 2869 MRS2S Fwd + MRS2 Rev MRS2S 957 1913 to 2869

The promoter corresponding to the cDNA 2D14 was designated as MRS3 (SEQID NO: 3). MRS3 was amplified out of Genome Walker library EcoRV by PCRwith primers 2D14DS1, 5′-ATGGGTTTGCGGGTATGGGTAGTGGTA-3′ (SEQ ID NO: 53)and 2D14GSP5, 5′-ACACCACCAGGTTCACGGCGAGGAACAG-3′(SEQ ID NO: 54). Attsites were added to MRS3 by PCR amplifying the product of the primaryreaction with primers consisting of ½ att and ½ gene-specific sequence,2D14DS3) 5′-ACAAAAAAGCAGGCTGCGGGTATGGGTAG-3′ (SEQ ID NO: 55) and2D14DS5, 5′-ACAAGAAAGCTGGGTTGCTCGATCACAACA-3′ (SEQ ID NO: 56). The fullattB1 and attB2 sites were added by using primers att1 (SEQ ID 43) andattB2 (SEQ ID NO: 44).

The promoter corresponding to the cDNA 4H19 was designated as MRS4 (SEQID NO: 4). MRS4 was amplified out of Genome Walker library DraI withprimers 4H19GSP3, 5′-TCTTTCTTGTGCACCGCCGAAGC-3′ (SEQ ID NO: 57) and4H19profor, 5′-ACCCGATAACGAGTTAACGATATGAACTGG-3′ (SEQ ID NO: 58)followed by amplification with nested primers 4H19GSP4,5′-TGCACTCACACCGCCGATGATGG-3′ (SEQ ID NO: 59) and 4H19profor (SEQ ID NO:58). Att sites were added to MRS4 by PCR amplifying the product of theprimary reaction with primers consisting of ½ att and ½ gene-specificsequence, 4H9proforB1, 5′-ACAAAAAAGCAGGCTAACGATATGAACTGG-3′ (SEQ ID NO:60) and 4H19 prorevB2, 5′-ACAAGAAAGCTGGGTCTTCACGAGTTCGGT-3′ (SEQ ID NO:61). Each promoter with attB1 and attB2 ends was recombined into plasmidpDONR 201(Life Technologies Cat. No. 11798-014), a donor vector withattP recombination sites, using the BP clonase mix of recombinationenzymes (Life Technologies Cat. No. 11789-013). In this entry vectoreach promoter was directionally cloned with the 5′ end of the promoteradjacent to the attL1 site and the 3′ end adjacent to the attL2 site. Aseparate entry vector was made for each of the four promoters.

Destination Vectors

A Binary destination vector, useful for plant transformnation, wasconstructed in the following manner. A recombination fragment (rf)containing lambda bacteriophage attachment-R (attR) sites flanking achloramphenicol resistance gene and a controlled cell death (ccd) genewas obtained from Invitrogen (Cat. No. 11828-019). Oligonucleotides wereobtained to amplify the rf cassette by PCR, incorporating a flanking 5′Bell site, rfBcII, 5′-TGCCCGTATGATCAACAAGTTTGTACAAAA-3′ (SEQ ID NO: 62)and a 3′ EcoRV site, rfEcoRV, 5′-CCAGACCGATATCAACCACTTTGTACAAGA-3′ (SEQID NO: 63) into the cassette. The addition of these unique restrictionsites made it possible to directionally subclone the recombinationfragment in front of the β-glucouronidase (GUS) gene and nopalinesynthase (nos) terminator (rf::gus::nos) to produce an intermediatedonor vector. The expression cassette (rf::gus::nos) was removed fromthe donor vector by endonuclease digestion using KpnI and gelpurification of the ˜3 kb fragment. A recipient binary vector (Negrottoet al., 2000 Plant Cell Reports 19: 798-803) was prepared for ligationof the expression cassette by endonuclease digestion using KpnI anddephosphorelated using calf intestine phosphatase (CIP). The expressioncassette was then subcloned into the KpnI site within the T-DNA region,near the right border of a binary plasmid. The T-DNA region of the finalbinary destination vector harbored the recombination expression cassettefollowed by a plant selectable marker expression cassette comprising themaize Ubiquitin promoter driving the phosphomannose isomerase gene andnos terminator (Negrotto et al., 2000 Plant Cell Reports 19: 798-803).The root-specific promoters, MRS1L, MRS1S, MRS2L, MRS2M, MRS2S, andMRS3, were each cloned into the final binary recombination vector infront of the GUS coding sequence. Thus, six binary vectors were made,MRS1L-GUS, MRS1S-GUS, MRS2L-GUS, MRS2M-GUS, MRS2S-GUS, and MRS3-GUS.

Example 6 Transient Expression in Maize Directed by Root-specificPromoters

Maize seeds were germinated and grown in the dark under sterileconditions for seven days. Root tissue was cut into I cm segments withsterile razor blades and arranged onto agarose plates prepared with2JMS+AG media (JMS majors, SH minors, MS iron, 2% sucrose, 5 mg/mldicamba, G5 additions, 10 mg/ml AgNO₃). Five micrograms of DNA fromvectors comprising MRS1L-GUS, MRS1S-GUS, MRS2L-GUS, and MRS3-GUS wasprecipitated onto (<1 μm) gold carriers and spotted onto micro-carriersfor bombardment at 650 psi as described (Wright et al. 2001, Plant CellReports, 20: 429-436). Fragment DNA was precipitated onto goldmicrocarriers (<1 μm) as described in the DuPont Biolistics Manual.Genes were delivered to the target tissue cells using the PDS-1000 HeBiolistics device. The approximate settings on the device were asfollows: 8mm between the rupture disk and the macrocarrier, 10 mmbetween the macrocarrier and the stopping screen and 7 cm between thestopping screen and the target. The plates were shot twice withDNA-coated particles using 650 psi-rupture disks. To reduce tissuedamage from the shock wave of the helium blast, a stainless steel mesh,with 200 openings per lineal inch horizontally and vertically(McMaster-Carr, New Brunswick, N.J.), was placed between the stoppingscreen and the target tissue. Roots were cultivated on the plates for 48hrs in the dark at 25° C. Expression of GUS was visualized by stainingwith 100 mM NaPO₄, 0.5 mM Potassium Ferricyanide, 0.5 mM PotassiumFerrocyanide, 10 mM EDTA pH 8.0, 0.5 mg/ml X-Gluc, 0.1% Triton X-100.Table 3 shows the relative levels of GUS expression of the root-specificpromoter constructs compared to a construct with the constitutive maizeubiquitin promoter driving expression of GUS, UbiP-GUS. A wildtype (Wt)maize plant was used as the negative control. GUS activity ischaracterized as high (+++), medium (++), low (+), or absent (−).Results indicate that promoters of the invention function in plant cellsand drive the expression of a heterologous coding sequence at comparablelevels to a constitutive promoter.

TABLE 3 Results of Transient Expression of GUS in Maize Roots. ConstructRelative GUS Expression Levels Wt − UbiP-GUS ++ MRS1L-GUS ++ MRS1S-GUS++ MRS2L-GUS ++ MRS3-GUS ++

Example 7 Expression of GUS in Stably-transformed Corn and Rice Directedby Root-specific Promoters

Maize and rice plants were transformed with expression cassettescomprising promoters of the invention operably linked to the GUS codingsequence. GUS activity in stably transformed maize and rice tissues wasmeasured by a fluorometric assay. Plant tissue was ground in extractionbuffer (50 mM Na₂HPO₄ pH7.0, 5 mM DTT, 1 mM NA₂ EDTA, 0.1% Sarcosyl,0.1% Triton-X). The reaction was carried out with approximately one parttissue extract to 5 parts assay buffer (extraction buffer with 47 mM4-methylumbelliferyl-glucuromide) at 37° C. and stopped with 2% Na₂CO₃at multiple time points. The activity was measured in a TecanSpectrafluor Plus at 365 nm excitation and 455 nm emission calibratedwith 4-MU standards diluted in Na₂Co₃. The slope of MU fluorescenceverses time gives the activity of the GUS enzyme. Protein concentrationswere measured by BCA assay (Pierce Cat. Nos. 23223 and 23224) and Gusactivity was normalized for protein concentration. Gus activity wascharacterized as high (+++), medium (++), low (+), or absent (−) anddata from 12 to 31 single copy transgenic maize plants or 7 single copytransgenic rice plants were averaged for each promoter construct.Results shown in Tables 4 and 5 demonstrate that GUS activity intransgenic plants comprising an expression cassette that comprises apromoter of the invention was confined specifically to the roots.

TABLE 4 GUS Expression in Tissues Excised from Transgenic Maize Plants.GUS Activity in Designated Maize Tissue Promoter Root Leaf Silk PollenKernel UbiP +++ +++ +++ +++ +++ MRS1L + − − − − MRS1S + − − − − MRS2L +− − − − MRS3 + − − − −

TABLE 5 GUS Expression in Tissues Excised from Transgenic Rice Plants.GUS Activity in Designated Rice Tissue Promoter Root Leaf Wildtype − −UbiP +++ +++ MRS2L +++ − MRS2M +++ − MRS2S +++ −

Example 8 Expression of Insecticidal Toxin Using Root-Specific Promoter

Maize plants were transformed with an expression cassette comprising theMRS3 promoter operatively linked to a heterologous coding sequence thatencodes a modified Cry3A toxin (U.S. application Ser. No. 60/316,421).Excised roots of transformed plants expressing the insecticidal toxinwere bioassayed against western corn rootworm and were observed to beinsecticidal. Transformed plants comprising the MRS3-modified Cry3Aexpression cassette were resistant to corn rootworm larval feedingdamage.

It will be recognized by those skilled in the art that the promoters ofthe present invention can be operably linked to other heterologouscoding sequences that encode toxins active against corn rootworm, forexample, the PS149B1 proteins (Moellenbeck et al. 2001, NatureBiotechnology 19: 668-672), a modified Cry3Bb toxin (U.S. Pat. No.6,063,597), the Diabrotica toxins disclosed in U.S. Pat. No. 6,281,413,or the Vip1-Vip2 toxin (U.S. Pat. No. 5,872,212).

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious to persons skilled in the art thatcertain changes and modifications may be practiced within the scope ofthe present invention.

1. An isolated nucleic acid molecule which codes for a promoter capableof directing root-specific transcription in a plant, wherein saidpromoter comprises the nucleotide sequence set forth in SEQ ID NO:
 3. 2.An expression cassette comprising, in sequence, the nucleic acidmolecule of claim 1 operably linked to a heterologous coding sequence,which is operably linked to a 3′-untranslated region including apolyadenylation signal.
 3. The expression cassette according to claim 2,wherein said heterologous coding sequence is selected from the groupconsisting of insecticidal coding sequences, nematicidal codingsequences, herbicide tolerance coding sequences, anti-microbial codingsequences, antifungal coding sequences, anti-viral coding sequences,abiotic stress tolerance coding sequences, nutritional quality codingsequences, visible marker coding sequences and selectable marker codingsequences.
 4. The expression cassette according to claim 3, wherein saidinsecticidal coding sequence encodes a toxin active against acoleopteran pest.
 5. The expression cassette according to claim 4,wherein said coleopteran pest is a species in the genus Diabrotica. 6.The expression cassette according to claim 3, wherein said visiblemarker is beta-glucuronidase.
 7. A recombinant vector comprising theexpression cassette of claim
 2. 8. The recombinant vector according toclaim 7, wherein said vector is a plasmid.